CN115025091B - Application of liensinine in preparation of drug for resisting African swine fever virus - Google Patents
Application of liensinine in preparation of drug for resisting African swine fever virus Download PDFInfo
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Abstract
The invention provides application of liensinine in preparation of a drug for resisting African swine fever viruses, and belongs to the field of antiviral application. Experiments prove that the liensinine shows obvious antiviral activity in vitro experiments, and can show extremely strong anti-African swine fever virus infection effect at the concentration of 5 mu M. The liensinine is a natural product extracted from traditional Chinese medicine lotus seeds, has the effects of reducing blood pressure, inhibiting smooth muscle contraction and the like, and has no toxic or side effect of obvious damage and the like on the physiological functions of organisms compared with hormone, antibiotic, chemical synthetic medicine and the like. Therefore, the liensinine has good prospect in preparing the African swine fever virus resistant medicament.
Description
Technical Field
The invention belongs to the field of antiviral application, and particularly relates to application of liensinine in preparation of a medicine for resisting African swine fever viruses.
Background
African Swine Fever (ASF) is an acute and virulent infectious disease caused by African Swine Fever Virus (ASFV) infection, and the lethality rate is up to 100%. ASF was first discovered in kenya in 1921 and then gradually spread to sub-saharan africa regions and over 60 countries and regions of europe, america, asia, etc. with increasing prevalence areas, is the first threat to the global swine industry. In 8 months in 2018, the ASF epidemic situation appears in China for the first time, and then the ASF epidemic situation rapidly spreads to the whole country, so that the economic benefit of the pig industry in China is seriously damaged.
ASFV is a nucleoplasmic giant DNA virus, belongs to the order of DNA viruses, the family of African swine fever viruses and the genus of African swine fever viruses, is the only member of the family, and is the only known arbovirus DNA virus at present. The ASFV virion has a double-layer envelope icosahedral structure and consists of an inner core, an inner core shell, an inner membrane, a capsid and an envelope, and the genome is a linear double-stranded DNA molecule with the size of 170-193 kb. The ASFV genome encodes 150-167 open reading frames, 68 structural proteins and more than 100 non-structural proteins.
Liensinine (LIEN), chemical structural formula is shown in figure 1, and molecular formula C 37 H 42 N 2 O 6 Is a dibenzyl tetrahydroisoquinoline alkaloid. Liensinine is an important functional compound in lotus seeds, has wide physiological activity and has the effects of inhibiting smooth muscle contraction, reducing blood pressure and resisting arrhythmia. The lotus seed serving as a common Chinese herbal medicine can clear heart and activate spleen, tonify spleen and stop diarrhea, nourish heart and tranquilize mind, improve eyesight, tonify middle-jiao and nourish mind, stop diarrhea and consolidate essence, tonify kidney and astringe essence and stop leucorrhea, and nourish primordial qi, and has practical application effects in clinic, but there are only few relevant reports in the field of antivirus.
The prevention and control of ASFV lacks effective vaccines and antiviral drugs, wherein the safe and effective antiviral drugs can effectively improve the survival rate of infected pigs, reduce economic loss and prevent the spread of viruses. On the premise that the research and development of the vaccine are trapped in the current predicament, the research and the development of the anti-ASFV drug have great significance for the prevention and the control of the epidemic situation of the ASFV.
Disclosure of Invention
The invention aims to solve the problem of epidemic African swine fever virus and provides application of liensinine in preparation of a drug for resisting African swine fever virus.
The purpose of the invention can be realized by the following technical scheme:
use of liensinine in preparing medicine for resisting African swine fever virus is provided.
A medicine for resisting African swine fever virus contains liensinine.
In the present invention, the medicament further comprises a pharmaceutically acceptable carrier.
Has the advantages that: experiments prove that liensinine shows remarkable antiviral activity in vitro experiments, and can show extremely strong anti-African swine fever virus (MOI = 1) infection effect at the concentration of 5 mu M. The liensinine is a natural product extracted from traditional Chinese medicine lotus seeds, has the effects of clearing summer heat, promoting diuresis, stopping bleeding and relieving epigastric distention and regulating qi, and has no toxic or side effect such as obvious damage to the physiological function of an organism compared with hormone, antibiotic, chemical synthetic medicine and the like. Therefore, the liensinine has good prospect in preparing the African swine fever virus resistant medicament.
Drawings
FIG. 1 is the chemical formula of liensinine.
FIG. 2 shows the cell viability of liensinine on PAM cells with the abscissa being liensinine concentration in μ M; the cell viability is plotted on the ordinate in%.
FIG. 3 is CC of liensinine on PAM cells 50 Fitting results of values, log on abscissa 10 (liensinine concentration); the cell viability is plotted on the ordinate in%. Wherein the liensinine concentration unit is μ M.
FIG. 4 shows the activity of liensinine, pulegone and kavain in inhibiting African swine fever virus infection on PAM cells measured by Western Blot method.
FIG. 5 shows the activity of liensinine in inhibiting African swine fever virus infection on PAM cells measured by Western Blot method. Wherein Mock is a control group, and 0.5. Mu.M, 1. Mu.M, 2.5. Mu.M and 5. Mu.M are intervention concentrations of liensinine in the corresponding samples of each lane.
FIG. 6 shows the IC of the Western Blot protein bands, which were analyzed by Image J software to calculate the inhibition rate at the corresponding concentration and fit 50 Numerical values. The abscissa is Log 10 (liensinine concentration), wherein liensinine concentration is in μ M; the ordinate represents the inhibition rate in%.
FIG. 7 shows the activity of liensinine in inhibiting African swine fever virus infection on PAM cells by fluorescent quantitative PCR, the abscissa is liensinine concentration in μ M, wherein the liensinine concentration of 0 is a control group; the ordinate is the relative transcript level of the ASFV-B646L gene (transcript level of the ASFV-B646L gene in the test well for liensinine intervention/transcript level of the ASFV-B646L gene in the control group).
FIG. 8 is a graph of the activity of liensinine in inhibiting African swine fever virus infection on PAM cells measured by the erythrocyte adsorption assay, with liensinine concentration on the abscissa in μ M, where liensinine concentration of 0 is a control group; the ordinate is the viral titer, log 10 (half amount of adsorbed erythrocytes).
Detailed Description
The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the following embodiments.
Test materials
All tests were performed in a Biological safety protection third-level laboratory (BSL-3) according to standard procedures. The African swine fever strain CN2018 is separated and stored by the Chinese animal health and epidemiology center; porcine Alveolar Macrophage (PAM) is prepared by washing aseptically extracted Porcine lungs with PBS; other used chemical reagents, real-time fluorescent quantitative PCR primers and kits were purchased from Novozam (Vazyme); liensinine, pulegone, and kavain are all available from Selleck.
Example 1 determination of the cytotoxicity of liensinine on PAM cells
PAM cells were counted and diluted at 1X 10 in DMEM medium containing 10% (volume percent) Fetal Bovine Serum (FBS) 5 The concentration per well was added to a 96-well plate, and the mixture was incubated at 37 ℃ and 5% CO 2 Culturing in an incubator, and discarding DMEM culture solution after PAM cells are completely attached (about 6-8 h). Adding 100 μ L of lotus plumule alkali solution (1 μ M, 5 μ M, 10 μ M, 25 μ M, 50 μ M, 100 μ M) prepared by DMEM culture solution as solvent into each well, and making three duplicate wells (marked as lotus plumule alkali treatment wells) at each concentration; setting a negative control hole of the cell hole which is not treated by the liensinine, and only replacing the liensinine alkali solution with 100 mu L of DMEM culture solution compared with the liensinine treatment hole; blank control wells were set, which contained no liensinine and cells, and only 100 μ L of DMEM medium. Subjecting 96 well plates to 37 ℃ and 5% CO 2 After incubation in an incubator for 24 hours, 10. Mu.L of CCK-8 (Cell Counting Kit-8, cell Counting reagent) was added to each well, and incubated at 37 ℃ for 1-2 hours. After the incubation is finished, reading the absorbance 0D at 450nm by using an enzyme-linked immunosorbent assay 450nm The cell survival rate (%) was calculated according to the following formula: cell survival rate = [ (As-Ab)/(An-Ab)]100 As is absorbance of the lotus plumule alkali-treated wells, an is absorbance of the negative control wells, ab is absorbance of the blank control wells, and the results are shown in FIG. 2. CC for fitting liensinine 50 The value (half cytotoxic concentration, which is the concentration required to exert a toxic effect on half cells) is shown in FIG. 3, and the C of liensinine on PAM cells can be seenC 50 The value was 29.22. Mu.M, and at liensinine concentrations less than 10. Mu.M, there was no significant cytotoxicity (cell viability > 90%) on PAM cells. Therefore, when the liensinine concentration is less than 10 μ M, there is no significant difference in the viability of the PAM cell group treated with liensinine from the untreated PAM cell group.
Example 2 assay of liensinine Activity for inhibition of African Swine fever Virus infection on PAM cells
This example examines the activity of liensinine, pulegone and kavain as well as different concentrations of liensinine on PAM cells to inhibit infection by african swine fever virus.
PAM cells were counted, diluted to an appropriate density with DMEM medium containing 10% (volume percent) Fetal Bovine Serum (FBS), and cultured at 2X 10 6 The concentration of each well was increased to 6-well plates, incubated at 37 ℃ and 5% 2 Culturing in an incubator, after the PAM cells are completely attached (about 6-8 h), discarding the DMEM culture solution, adding 2mL of the DMEM culture solution (control hole), 5 mu M lotus plumule alkali solution, 10 mu M pulegone solution and 10 mu M kavain solution into each hole respectively, pretreating the PAM cells for 2h, discarding the culture solution in the holes, and then adding 2mL of the DMEM culture solution (control hole) and a suspension which is prepared by taking the DMEM culture solution as a solvent and contains the drug and the African swine fever CN2018 strain (the type and the concentration of the drug in each hole are consistent with those in pretreatment), wherein the virus inoculation amount in each hole is MOI =1. Placing 6 well plates at 37 ℃ and 5% CO 2 After culturing for 24h in an incubator, collecting cell samples, detecting the expression condition of p72 protein by Western Blot, taking beta-actin protein as internal reference, wherein primary antibodies are ASFV p72 protein mouse monoclonal antibody (purchased from Toyobo Co., ltd.) and beta-actin protein rabbit polyclonal antibody (purchased from Proteintetech, with the product number of 20536-1-AP), and secondary antibodies are horseradish peroxidase labeled goat anti-mouse IgG antibody (purchased from Bioworld, with the product number of BS 12478) and horseradish peroxidase labeled goat anti-rabbit IgG antibody (purchased from Bioworld, with the product number of BS 13278), respectively, and as a result, as shown in FIG. 4, the liensine can obviously inhibit the replication of African fever swine fever virus compared with other drugs and controls.
PAM (polyacrylamide) cytometerSeveral times, diluted to appropriate density with DMEM medium containing 10% (volume percent) Fetal Bovine Serum (FBS), and then diluted at 2X 10 6 The concentration per well was measured in 6-well plates, incubated at 37 ℃ and 5% CO 2 Culturing in incubator, adding 100 μ L DMEM culture solution (control group) and lotus plumule alkali solution (experimental group) with concentration of 0.5 μ M, 1 μ M, 2.5 μ M and 5 μ M prepared by using DMEM culture solution as solvent into each well after the cells are completely attached (about 6-8 h), pretreating PAM cells at 37 deg.C for 2h, inoculating African swine fever virus CN2018 strain into each well according to MOI =1, placing at 37 deg.C and 5% CO 2 Adsorbing for 1h in the incubator, removing the culture solution in the wells, washing twice with PBS buffer solution, adding 2mL of DMEM culture solution (control group) or 0.5. Mu.M, 1. Mu.M, 2.5. Mu.M and 5. Mu.M plumula Nelumbinis alkali solution prepared with DMEM culture solution as solvent (concentration of plumula Nelumbinis alkali in each well is the same as that in pretreatment), placing at 37 deg.C, and adjusting to 5% CO 2 After culturing for 24h in an incubator, collecting cell samples, detecting the expression condition of the p72 protein by Western Blot, taking beta-actin protein as internal reference, wherein the primary antibodies are ASFV p72 protein mouse monoclonal antibody (purchased from Toyowa bioscience) and beta-actin protein rabbit polyclonal antibody (purchased from Proteitech, with the product number of 20536-1-AP), and the secondary antibodies are horseradish peroxidase labeled goat anti-mouse IgG antibody (purchased from Bioworld, with the product number of BS 12478) and horseradish peroxidase labeled goat anti-rabbit IgG antibody (purchased from Bioworld, with the product number of BS 13278). The results are shown in FIG. 5. Analyzing the gray value of the protein band by using Image J, obtaining the gray value of a target band (p 72 protein)/gray value of an internal reference band of each lane, and calculating the inhibition rate of liensinine on ASFV under each concentration, wherein the formula is as follows: inhibition =1- [ (target band gray-value of experimental group/reference band gray-value of experimental group)/(target band gray-value of control group/reference band gray-value of control group)]And fitting IC of liensinine according to inhibition rate 50 (inhibition concentration 50) value, IC 50 Is the median inhibitory concentration, which means the liensinine concentration that inhibits half of the ASFV. As can be seen from FIG. 6, IC of liensinine 50 =1.512 μ M. Selectivity index SI (SI) = CC 50 /IC 50 The greater the value of SI, the inhibition of the drugThe stronger the use, the higher the safe concentration. The SI value of the liensinine is 29.22/1.512=19.33, and is far larger than that of other reported anti-ASFV drugs.
Example 3 fluorescent quantitative PCR assay of liensinine Activity for inhibiting African Swine fever Virus infection on PAM cells
After PAM cell count, the cells were diluted to an appropriate density with DMEM medium containing 10% (volume percent) Fetal Bovine Serum (FBS), and then diluted to 2X 10 6 The concentration per well was measured in 6-well plates, incubated at 37 ℃ and 5% CO 2 Culturing in incubator until the cells are completely attached (culture time is about 6-8 h), removing the culture solution, pretreating PAM cells at 37 deg.C for 2h with 2mL DMEM culture solution (control group) and 5 μ M plumula Nelumbinis alkali solution prepared with DMEM culture solution as solvent, inoculating African swine fever CN2018 strain according to MOI =1, placing at 37 deg.C, and culturing at 5% CO 2 Adsorbing for 1 hr in incubator, removing culture medium in the wells, washing with PBS buffer solution twice, adding 2mL of DMEM culture solution (control group) or 5 μ M plumula Nelumbinis alkali solution prepared with DMEM culture solution as solvent (the concentration of plumula Nelumbinis alkali in each well is consistent with that in pretreatment), placing at 37 deg.C, and reacting with 5% CO 2 After 24 hours of culture in an incubator, a cell sample was collected by TRIzol (total RNA extraction reagent, purchased from Solarbio), RNA was extracted, the RNA was reverse-transcribed into cDNA according to a conventional method, and a change in mRNA level of the p72 protein of ASFV, i.e., a change in transcription level of the ASFV-B646L gene, was detected by real-time fluorescent quantitative PCR using the Actin gene as an internal reference. The PCR amplification primers of the Actin gene comprise Actin-F and Actin-R, the PCR amplification primers of the ASFV-B646L gene comprise ASFV-B646L-F and ASFV-B646L-R, and the specific sequences of the primers are as follows:
Actin-F：5'-CACGCCATCCTGCGTCTGGA-3'；
Actin-R：5'-AGCACCGTGTTGGCGTAGAG-3'；
ASFV-B646L-F：5'-GTGGTGAGTGGGCTGCATAA-3'；
ASFV-B646L-R：5'-TAAAACCTACCTGGAACATCTCC-3'。
wherein the PCR reaction system comprises: 10 μ L of AceQ qPCR SYBR Green Master Mix (High ROX premix), 0.4 μ L of forward primer (10 μ M), 0.4 μ L of reverse primer (10 μ M), 2 μ L of template DNA (i.e., cDNA obtained by reverse transcription), and 7.2 μ L of sterile distilled water. The PCR reaction program is: pre-denaturation at 95 ℃ for 5min; 10s at 95 ℃ and 30s at 60 ℃ for 40 cycles. And (3) measuring a dissolution curve after the reaction is finished, wherein the reaction conditions are as follows: 95 ℃ for 15s, 60 ℃ for 60s and 95 ℃ for 15s. Wherein the AceQ qPCR SYBR Green Master Mix (High ROX Premixed) is purchased from Nanjing Nodezam Biotech GmbH, having a product number of Q141-02.
This experiment was carried out by 2 -ΔΔCt The method carries out relative quantification of ASFV-B646L gene, the result is shown in figure 7, which shows that liensinine obviously inhibits the activity of African swine fever virus infection on PAM cells, and has obvious difference with a control group (the method has the advantages of obvious inhibition effect on the activity of the liensinine on the activity of the PAM cells, and the method has the advantages of high safety, high safety and high safety (the method has the advantages of high safety and high safety)P＜0.0001）。
Example 4 erythrocyte adsorption assay (HAD) determination of liensinine Activity on PAM cells to inhibit infection by african swine fever virus
After PAM cell counting, the cells were diluted to an appropriate density with 10% (volume percentage concentration) Fetal Bovine Serum (FBS) in DMEM medium and then diluted to 1X 10 5 The concentration per well was measured in a 96-well plate, at 37 ℃ and 5% CO 2 Culturing in incubator until cells are completely attached (about 6-8 h), removing culture solution, adding 100 μ L DMEM culture solution (control group) and 5 μ M plumula Nelumbinis alkali solution (test group) prepared with DMEM culture solution as solvent into each well, pretreating PAM cells for 2h, and mixing with stock solution of ASFV CN2018 strain (1 × 10) 7 HAD 50 /mL) at a dilution of 10 -1 、10 -2 …10 -8 Diluting the concentration gradient, and respectively adding virus liquid with each concentration into a control group and an experimental group of a 96-well plate according to 100 mu L/well, wherein each concentration is set to be 8 times; after 1 hour of ASFV virus adsorption, the well-cultured solution was discarded, PBS buffer was washed twice, 100. Mu.L of DMEM medium was added to each well of the control group, 100. Mu.L of a 5. Mu.M plumula Nelumbinis alkali solution (solvent is DMEM medium) was added to each well of the test group, 25. Mu.L of a 1% pig red blood cell suspension was added to each well, the mixture was allowed to stand at 37 ℃ and 5% CO 2 Culturing in incubator for 7-10 days, observing erythrocyte adsorption condition under microscope day by day, counting cell holes with rosette or mulberry shape formed by attaching a large amount of pig erythrocytes around cells, and calculating HAD by Karber method 50 (half-reddish fineness)Cell adsorption amount), the results are shown in figure 8, liensinine can obviously inhibit the African swine fever virus infection, and the virus titer of the test group is 1 multiplied by 10 compared with the control group 7 HAD 50 the/mL is reduced to 1 × 10 3.67 HAD 50 mL, significant difference between groups: (A), (B), (C)P＜0.0001）。
Claims (2)
1. Use of liensinine in preparing medicine for resisting African swine fever virus is provided.
2. The use according to claim 1, characterized in that said liensinine is added with a pharmaceutically acceptable carrier.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009150281A1 (en) * | 2008-06-12 | 2009-12-17 | Consejo Superior De Investigaciones Científicas | Use of lauryl gallate in the prevention and treatment of infections caused by african swine fever virus (asfv) |
| CN110786414A (en) * | 2019-12-03 | 2020-02-14 | 六安恒佳生物科技有限公司 | Fermented traditional Chinese medicine additive for preventing African swine fever and preparation method thereof |
| CN111228428A (en) * | 2020-04-08 | 2020-06-05 | 杨作丰 | Liquid traditional Chinese medicine preparation for preventing African swine fever and method thereof |
| CN112691105A (en) * | 2020-07-02 | 2021-04-23 | 中国人民解放军军事科学院军事医学研究院 | New use of neferine in inhibiting SARS-CoV and SARS-CoV-2 infection |
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| RU2020131755A (en) * | 2018-03-02 | 2022-04-04 | Канзас Стейт Юниверсити Рисеч Фаундейшн | CHEMICAL REDUCTION OF AFRICAN SWINE FEVER VIRUS AND CLASSICAL SWINE FEVER VIRUS |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009150281A1 (en) * | 2008-06-12 | 2009-12-17 | Consejo Superior De Investigaciones Científicas | Use of lauryl gallate in the prevention and treatment of infections caused by african swine fever virus (asfv) |
| CN110786414A (en) * | 2019-12-03 | 2020-02-14 | 六安恒佳生物科技有限公司 | Fermented traditional Chinese medicine additive for preventing African swine fever and preparation method thereof |
| CN111228428A (en) * | 2020-04-08 | 2020-06-05 | 杨作丰 | Liquid traditional Chinese medicine preparation for preventing African swine fever and method thereof |
| CN112691105A (en) * | 2020-07-02 | 2021-04-23 | 中国人民解放军军事科学院军事医学研究院 | New use of neferine in inhibiting SARS-CoV and SARS-CoV-2 infection |
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